Sorafenib Can Inhibit PI3K/AKT And MAPK Pathway Remarkably Activated By Rapamycin In Hepatocellular Carcinoma

Sorafenib can Inhibit PI3K/AKT and MAPK Pathway Remarkably Activated by Rapamycin in Hepatocellular CarcinomaHepatocellular carcinoma (HCC) is one of the most common cancers in China in terms of number of cases. As an important and effective therapeutic tool, liver transplantation for HCC has been applied widespread and gotten encouraging therapeutic effects. However,tumor recurrence and metastasis after liver transplantation has been the major reason to compromise long-term-survival of patients. Developing more effective tools may conduce to increased therapeutic effects for preventing or treating the recurrence and metastasis after liver transpantation for HCC.For the past few years, more and more molecularly targeted agents have gotten encouraging therapeutic effects in many cancers, including HCC. Targeted the overactive signalling molecules, association with tumor growth, proliferation, invasion, metastasis, angiogenesis and so on, may be a potentiality instrument for preventing or treating the recurrence and metastasis after liver transpantation for HCC. Rapamycin, a mTOR inhibitor, has been effectively applied for anti-rejection after liver transplantion. Meanwhile, the antitumoral effects of rapamycin had been discovered in many fundamental and clinical researches. However, drug fast of rapamycin has been the major barrier for clinic antitumoral application. Developing other therapeutic tools combined with it may effectively integrate the dual anti-rejection and antitumoral effects of rapamycin,this will be a possible train of thought to prevent and treat the recurrence and metastasis after liver transpantation for HCC.Sorafenib,a mutikinase inhibitor,can be applied for antitumor safely and effectively contributed to its Raf and RTKs(Receptor tyrosine kinase) inhibitory activities, including in advanced HCC. In this article, we investigate the drug fast mechanism of rapamycin and further to know how sorafenib improves this resistance in HCC. In our study, we find out the phenomenon that rapamycin can't inhibite HCC cell lines growth very effectively in vitro, and this may contribute to paradoxically up-regulated of AKT and ERK activation by rapamycin. sorafenib can inhibite PI3K/AKT and MAPK pathway remarkably activated by rapamycin and enhance growth inhibition in vitro; at last,we confirm this synergism in a potential liver transplantation rat model of HCC in vivo.Part One Growth inhibitory effects of rapamycin in human HCC cell lines Objective:To investigate the growth inhibitory effects of rapamycin in HCC cell lines.Methods:Two HCC cell lines, HCC-LM3 and HepG2, were treated with rapamycin at different concentrations(0-100ng/ml) for different periods(24h,48h,72h), then MTT was used to detect the growth inhibitory effects.Results:Rapamycin could inhibite growth of both HCC-LM3 and HepG2 cell line, however, growth inhibitory effects of rapamycin were not satisfactory, inhibitory rates to HCC cell lines were under 35% (HepG2 28.4%; HCC-LM3 34.1%),even though treated with rapamycin at high concerntration and long time (100ng/ml,72h).Conclusion:Growth inhibitory effects of rapamycin in HCC cell lines were not very satisfactory, suggested that HCC may be refractory to rapamycin.Part Two PI3K/AKT and MAPK Pathway abnormally Activated by Rapamycin in human HCC cell linesObjective:To investigate refractory mechanism of rapamycin in human HCC cell lines.Methods:HCC-LM3 and HepG2 cell lines were treated with rapamycin at different concentrations for 2 hours or at lOng/ml for different periods, cell proteins were collected and Western blot was used to detect the phospho-AKTser473,phospho-ERK1/2 and PDGFR-βlevels. HCC cell lines were pretreated with LY294002, a PI3K inhibitor, then co-treated with rapamycin(50ng/ml) and LY294002 for 2 hours, Western blot was also used to detect the phospho-AKTser473 and phospho-ERK1/2 levels. HCC cell lines were treated with rapamycin at 10ng/ml for 24 hours, cells were collected and RNAs were abstracted,then qRT-PCR was used to detect the expression levels of PDGFR-a and PDGFR-β.Results:Rapamycin had no effect on the total-AKT and total-ERK expression. Rapamycin could paradoxically up-regulate AKT activation quickly and persistently, also in a time and dose dependent manner. Rapamycin didn't up-regulate phospho-ERK1/2 level obviously at lOng/ml for 2 hours, while at higher concentrations for longer periods, phospho-ERK1/2 levels were paradoxically up-regulated obviously in a time and dose dependent manner. LY294002 could down regulate phospho-AKTser473 and phospho-ERK1/2 levels in HCC cell lines. Rapamycin didn't up-regulate AKT activation, but phospho-ERK1/2 levels could still be up-regulated when pretreated with LY294002. Rapamycin could rise PDGFR-βexpression in HCC-LM3 and HepG2 cell lines, significantly in HepG2 cell line.Conclusion:Rapamycin could paradoxically activate AKT and ERK in a time and dose dependent manner. Activated AKT by rapamycin depended on PI3K, however, upregulation of phospho-ERK1/2 levels may partly depend on PI3K. The expression of PDGFR-βupregulated by rapamycin may be an important reason for paradoxically up-regulated AKT and ERK activation.Part Three Sorafenib can Inhibit PI3K/AKT and MAPK Pathway Remarkably Activated by Rapamycin in vitroObjective:To investigate inhibitory effects of sorafenib for PI3K/AKT and MAPK pathway paradoxically activated by rapamycin.Methods:HCC-LM3 and HepG2, were treated with sorafenib alone or in combination with rapamycin at different concentrations for different periods, then MTT was used to detect the growth inhibitory effects. These two cell lines were treated with sorafenib alone or in combination with rapamycin at default concentrations for different periods, cell proteins were collected and Western blot was used to detect the phospho-AKTser473,phospho-ERKl/2 and PDGFR-βlevels. HCC cell lines were treated with sorafenib alone or in combination with rapamycin for 24 hours, RNAs were abstracted and qRT-PCR was used to detect the expression level of PDGFR-p.Results:Combination with sorafenib could enhance growth inhibitory effects of rapamycin strikingly in HCC-LM3 and HepG2 cell lines, also in a time and dose dependent manner. Soarafenib alone could inhibite ERK activation strikingly in a time and dose dependent manner.AKT activation also could be inhibited by sorafenib although only in partial time and dose dependency. Combinated with sorafenib could strikingly inhibite AKT and ERK activation in a time and dose dependent manner, even at low concentration (2μM) for 2 hours. qRT-PCR and Western blot showed that sorafenib could down regulate PDGFR-βexpression remarkably both at basal level and abnormal up-regulated level.Conclusion:Growth inhibitory effects of rapamycin were strikingly enhanced by sorafenib. Sorafenib alone could inhibite AKT and ERK activation.Combination with sorafenib could remarkably inhibite AKT and ERK activation which were paradoxically up-regulated by rapamycin. These effects may be contributed to down-regulated PDGFR-βexpression and activation by sorafenib.Part Four Antitumoral effects of rapamycin alone and in combination with sorafenib in a rat HCC cell line and in a potential liver transplantation rat model of hepatocellular carcinoma Objective:To investigate antitumoral effects of rapamycin alone and in combination with sorafenib in MH3924A cell line and in an orthotopic rat model of hepatocellular carcinoma.Methods:MTT assay was used to detect growth inhibitory effects of rapamycin alone or in combination with sorafenib in MH3924A cell line. Tumor cells were treated with rapamycin alone or in combination with sorafenib at default concentrations for different periods, at appropriate moments were pretreated with LY294002; Western blot was used to detect the phospho-AKTser473 and phospho-ERK1/2 levels. Cells were treated with rapamycin alone or in combination with sorafenib for 24 hours, RNAs were abstracted and qRT-PCR was used to detect the expression levels of Pdgfr-a and Pdgfr-β.Orthotopic model of hepatocellular carcinoma was performed on ACI rats. The rats were randomly divided into four groups: rapamycin group, sorafenib group, combination therapy group,and vehicle group. Tumor growth and volumes were monitored by color ultrasonography. Tumor volumes, tumor metastasis, tumor necrosis and tumor cell proliferation rates were compared after treatments. Tumor proteins were collected and Western blot was used to detect the phospho-AKTser473 and phospho-ERK1/2 levels.Results:In MH3924A cell line, Growth inhibitory effects of rapamycin were not very satisfactory and in combination with sorafenib could enhance growth inhibitory effects strikingly in time and dose dependency. Rapamycin could paradoxically up-regulate AKT and ERK activation remarkably;Activated AKT by rapamycin depended on PI3K, however, upregulated phospho-ERKl/2 levels may partly depend on PI3K. Combination with sorafenib could remarkably inhibite AKT and ERK activation which were abnormally up-regulated by rapamycin. qRT-PCR showed that sorafenib could down regulate PDGFR-βexpression remarkably either at basal level or at abnormal up-regulated level. Abnormal Pdgfr-a expressiont up-regulated by rapamycin may also be down regulated. In vivo, rapamycin or sorafenib alone inhibited tumor growth effectively, but rapamycin alone didn't inhibit tumor lung metastasis and abdominal lymph node metastasis effectively.Coadministration of rapamycin and sorafenib significantly enhanced the antitumoral effects on primary tumor growth and metastasis compared with single treatment. Additionally,the combination therapy further enhanced tumor necrosis and showed a significant inhibition of tumor cell proliferation. Combination with sorafenib could also remarkably inhibite AKT and ERK activation abnormally up-regulated by rapamycin in vivo.Conclusion:Combination therapy of rapamycin and sorafenib enhanced the antitumoral effects in vitro and in a rat model of HCC.These benefits may be closely related to inhibitory effcts of sorafeinb on AKT and ERK activation paradoxically upregulated by rapamycin.